Purification of dehydrocorticosterone acetate



Patented Mar. 30, 1948 BQB'IEICATION F DEHYDROCOR'DI- 'i QS EBQ ACETATE Stewart M. 'Miller 'and Jacob van-dc Kamp, Westfield,iN'.I;I.,. assigncrs to Merck .8; Co'.,LInc.,"Ral1- way,I'N. J.',"a corporation of New Jersey NqDrawing. Application April 13,1946,

"'fSerialNo. 662,105

This invention relates to the recovery'and isolation of dehydrocorticosterone acetate 'in an improved state of purity from reactionlmixtures containing this hormonai substance. More .particularly, our invention is-ccn'cerned with a new and improved process for the recovery of:3;I1-,20- triketo-A -21-acetoxypregnene (dehydrocorticosterone acetate) fromreaction mixturs containing this product by a procedure which permits the obtainment -of the hormonal substance in a higher state of purity and in-considerably higher yields than previously possible with the recovery processes now known in the. art.

Dehydrocorticosterone, tone of :the important adrenal cortex hormones, has heretofore been available-only in veryxminutetquantities because of the difficulty ofprep'arin'g'itheihormone in any substantial amount from ithe limited natural sources available. In attempts to render the hormone available in greater amounts, the prior art has developed a method for synthesizing the hormone in which it is obtained "in the form'of its acetate, 3,11,20-triketo-A zl acetoxypregnene', at the end of a long procedurelstaxrting with a relatively abundant naturally-occurring substance." This synthesis has been unsatisfactory, however, in-that the final step has given such low yields of the-desired :hormonal substance, dehydrocortico'stero'ne acetate, that the preparation of the hormone "by synthesis :has been economically impractical.

The primary object of-our invention is to provide an" improved procedure for the isolation and purification of dehydrocorticosterone' acetate from the crude reactionmixture in which it is pr duced'as-aresult of thiss'ynthesis. Thisproduct, 3,1lQZO-triketo-NfigZl acetoxypregnene, is theresult of a'diflicult, tedious, and costly procedure involving-a lar'g'enumb er of chemical reacti ons-p f intricate character; Th'eprincipal aim of-pur inventicri is to make-it possible to recover dehydrocortico'slterone acetate in 'a higher degree of purity and in better yields than previously possible, thereby greatly improving zthe synthetic process-as a means ofsecuring the pure hormonal substance ineconomically "feasible amounts.

InIth'e synthesis of dehydrocortico'ster'one .acetate (3, 11,20-triketo A 21 acetoxypregnene) as described in the literature by La-itdori andReichstein, Helvetica Ch'emlca'Acta126, 747-755 (.1943) there is obtained a crude reaction mixture 'by debrominating '3,ILZO-triketo-Aebromo-zl-acetoxypreg nane by refluxing thBAHbTOmO compound in pyridine. By this procedure dehydrocorticosterone acetate is secured. this .diesired product being contaminated, "however, with a multitude of impurities whichlai'e.extremely'difiicultto r move. .Lardon .and-Reich'stein have described a prdce durewhereinwatler soluble; basic, and acidic compounds'iofflthe gr t' 7 ix urem taken into acccuntjthesea s t nLproceedii r i separationlby means... .chrom graphic methods. 'At this stage, 1. .81, jffi qnt separation of thehighlyt comme x. reaction I ixture -d not occur. V

'We have found .that the impurities, which are present ong with the enytmcenicc ta ne acetate in the reaction mixture, tajiiter removal of .the water-soluble, basic 'anclacidtccompourrds, may be classed into various groupsfhamong which are theihigher molecular wei lit condensation products and comma; melting .crystalli'zable' po ents may. be was to a greater or less extent. In "our improved procedure for recovering iiehydrocorticosterone acetate'in high yield and infthe ub'stantiallypure state, we first extract the -ac andf'bja silc contaminents, as (in theprior art,

, ut iethen p 70,- c'eed to m ve the Q fl im u ities rese whichimpurities must he ."glim d if gthes ubstantially pure hormonal subs P is to be obtained in high yields.

vIn the recovery and purification procedure described in the literature, after debrominationbf 3,11,2 tr ket0-ifl-1b 9 someon "th reaction mixture is e ted-under reduced pressure to remove the pyridine; The residue is then taken up in alarg'eyqlumeof ether, and. w ed s ve y with 'si h ie'hvd hmr acid, with sodium carbo' ate' dilution and with wa It s hn' i q.'l'1" 51 Su ph e an concentrated. The resulting i ioiiuct' is chromatographed over a columnof'alumina. Upon elution with benzene; th' 'flrsflben ene 'eluates give crystals which' rnelt "relatively 1 w 'teniperature without a sharmelting point. The later benzene eluates, a be nefether eluates, furnish substantiallyco 7 stals melting at 1'78','but thepmquc ob aind-only in' low ields, and in none too -sa,tisfac"tcry e; stateiof purity.

We have found. that :thegprocedure -described in the prior art :for the recovery 50f dehydroe corticosterone acetate irom-the crude reaction mixture obtained by debromination of 3,1 1,20- triketo-4-bromo-2l acetoxypnegnane doesnot effectively remove the complex mixtures 'of impurities present thereinsnor. .068.113 yield. de ydlfqcorticosterone ,acetate in a suffi en ly hi the impurities, during the successive recrystallization, washing and other purification steps.

In our improved procedure, wherein the specific types of impurities present along with the dehydrocorticosterone acetate are removed by 7 than atmospheric, and the residue taken up in specific procedures designed to obtain maximum recovery of products, we areable to secure the product in high yields and in a high state of purity. Our procedure permits the removal of those impurities which may be classed as the higher molecular weight condensation products and complexes, oils and tars, and low-melting crystallizable mixtures, all of which impurities aremore or less dark in color. If left in the hormone, not only would its purity be much reduced, but they would greatly darken its color.

In our improved procedure we follow the purification method described in the chemical literature-to the extent that we first extract acidic and basic impurities present with the dehydrocorticosterone acetate in the reaction mixture. The crude neutral residue, obtained after evaporation of-the solvent in which the product is held in solution during the extraction procedure,

is then treated in accordance with out improved Example A solution of 10.0 grams of 3,11,20-trlketo-4- bromo-21-acetoxypregnane (melting point 185.6- 186.3 C. corrected) in 250 milliliters of redistilled pyridine was refluxed for 5. hours. I The pyridine was then removed under reduced pressure, less 146 milliliters of chloroform. The resulting solution was first washed with 292 milliliters of water, then with 584 milliliters of 1 N hydrochloric acid, and .then with 584 milliliters of a saturated solution' of sodium bicarbonate. Finally it was Washed with 292 milliliters of water.

The chloroform layer separating was concentrated to dryness and the residue was then taken up in 38 milliliters of benzene. Impurities present in the benzene solution were then precipitated by adding 740 milliliters of absolute ether, and allowing the resulting iiocculent precipitate to settle in the solution by standing for a period of several days.

The precipitate was then removed by filtration through Supercel filtration aid, and the filtrate weight of the dry crude material at this point method. This residue is dissolved in benzene and, upon the addition of ether, the higher molecular weight condensation products and complexes are throwndown in the form of a fiocculent precipitate. After removal of the precipi: tate by filtration, decantation, centrifugation, or otherwise, the filtrate, which contains the desired dehydrocorticosterone acetate product, is evaporated to dryness. j This dry product is next treated to free it from oily and tarry impurities. "This is accomplished by washing the residue with ether, and then. with a mixture'of acetone and ether. This treatment removes nearly all colored and lowmelting crystallizable materials. The, final traces of color in the product are removed by adsorption on activated alumina, the product being treated in the form of a solution in etherchloroform. After removing the solvent, recrystallization froma mixture of methanol and water in the presence of, a small amount of 7 ac-, tivated charcoal to remove any traces of impurities which may still remain results in pure, white crystals of dehydrocorticosterone acetate melting at l31-182.9 C. The product has a specific rotation-of (oz) 226i3. The yield is 32.6%based on the amount of 3,11,20-triketo- 4-bromo-2l-acetoxypregnane which was initially debrominated by refluxing in pyridine.

-Hormonal material.prepared in this way has been found to be identicalwith'dehydrocorticific rotation, carbon and hydrogen analyses, and

by the ultra-violet adsorption spectrum, and

bioassay.

an example of our new improved procedure 'forrecovering dehydrocorticosterone acetate in high yield and in a very high state of purity Y from reaction mixtures containing this hormone, i the following may be given.

was 3.80? grams or 46% of the theoretically possible yield.

The material was next transferred 'to a 30 milliliter centrifuge tube and slurried in 4 milliliters of acetone. To the mixture there was then added 8 millililtersof absolute'ether. The mixture was stirred until all lumps were reduced. The liquorswere then removed by centrifuging and decanting. Three more washes, each with 9"milliliters of absolute ether, were given the material. (38.5% of the theoretically possible yield).

The material was nextdissolved in a mixture of equal amounts of chloroform and absolute ether, and. run through a 6 centimeter column of activatedalumina' which was '1 centimeter in diameter. More of the solvent mixture was kept draining through the column until practically no more crystallizable material was found in the eluate. milliliters of the .mixture. The eluate was evaporated to dryness, leaving 'a' residue weighing 2.982 grams (36%of the theoretical). The melting point of'the product was;1'l9.2-l82 C. cor rected, with slight previous sintering.

. The. residue was dissolved in 224 milliliters of methanol, filtered, and 510 millililters of hot water added thereto. The'mixture was chilled and seeded when it began to cloud. The product crystallized rapidly in fine needles; It was allowed to stand in the refrigerator overnight before filtering. The filter cake was then Washed Various changes and modifications may be 7 made in our improved procedure, as described, without departing from'the spirit and scope of the invention. These changes and modifications, to the extent that they arewithin the scope of the appended'claims, are intended to be a part of our invention.

The 7 Its weight was now 3.200 grams 7 The entire operation required about 400 V We claim:

1. The process of securing dehydrocorticosterone acetate, which comprises: treating a reaction product containing dehydrocorticosterone acetate in order to remove water-soluble basic and acidic impurities present therein; concentrating the resulting crude partly purified neutral residue; dissolving the crude residue in benzene; adding ether thereto in order to precipitate the higher molecular weight condensation products and complexes present as impurities therein; removing said higher molecular weight condensation products and complexes; evaporating the resulting solution containing dehydrocorticosterone acetate in substantially purified form to dryness; removing oily and tarry impurities present therein by washing the residue first with ether, and then with a mixture of acetone and ether; adsorbing colored impurities present therein by treating an ether-chloroform solution of said purified products on activated alumina; and crystallizing said product in the form of pure white crystals of dehydrocorticosterone acetate.

2. The process of purifying a reaction mixture containing crude dehydrocorticosterone acetate in order to recover the said product in substantially pure form which comprises: dissolving the crude reaction mixture in chloroform; washing the resulting solution successively with water, hydrochloric acid and sodium bicarbonate solution; concentrating said washed chloroform solution to dryness; dissolving the dry residue in benzene; adding to the resulting benzene solution absolute ether in order to precipitate impurities; concentrating said benzene ether solution to dryness; treating said dry residue with absolute ether; washing said residue With a mixture of acetone and ether, and with ether alone; dissolving the residue in a mixture of chloroform and absolute ether; adsorbing impurities from said solution on activated alumina; evaporating the resulting purified solution to dryness; dissolving the resulting dry residue in methanol; adding hot water to said methanol solution; adsorbing impurities present in said resulting methanol-water solution by treatment with decolorizing carbon; and cooling said resulting solution in order to crystallize out therefrom dehydrocorticosterone acetate in the substantially pure state.

STEWART M. MILLER.

JACOB VAN DE KAMP.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,166,877 Reichstein July 18, 1939 2,238,868 Cartland Apr, 15, 1941 2,403,683 Reichstein July 9, 1946 2,404,768 Reichstein July 23, 1946 OTHER REFERENCES Ser. No. 202,126, Laqueur (A. P. C.) published Apr. 20, 1943. 

